Aim 1. Define the role of miRNAs in the regulation of transcriptional heterogeneity. MicroRNAs regulate gene expression by decreasing the stability of their target mRNAs. Although rules of miRNA targeting are well established, we and others have shown that even lethal phenotypes caused by the deletion of a miRNA gene are accompanied by only minor changes in target levels when measuring gene expression in a population of cells. Thus, it remains unclear how their regulatory activity impacts cellular behavior and contributes to human disease. One hypothesis is that in animals, the primary role of miRNAs is to regulate transcriptional noise, ensuring the faithful recapitulation of gene expression programs. Stochastic variation in gene expression plays an important role in cell fate decisions and has been implicated in a variety of other biological processes including survival of tumor cells to chemotherapy. I will test this hypothesis by characterizing the consequences of miRNA loss within individual cells of a population during early cell fate decisions and tumorigenesis, two settings in which miRNAs have been shown to play essential roles. Aim 2. Define the roles of the endogenous RNAi pathway in mammals. Mammals have four Argonaute proteins (Ago1-4), all of which are assumed to perform redundant roles in the miRNA pathway: once bound to a miRNA, these proteins are directed to the 3'UTR of target genes via a short complementary sequence (typically 6-8 nucleotides long) and recruit a large protein complex (RISC) that promotes target repression through mRNA destabilization and translational inhibition. In addition to this function, Ago2 also has a conserved catalytic domain, and can cleave targets that have extensive complementarity (20 nucleotides) to an Ago2-bound small RNA. This cleavage activity is essential in mammals. In fact mice expressing a catalytic dead Ago2-which remains competent to act in the miRNA pathway-die during embryogenesis, and deletion of Ago2 in the female germline leads to infertility due to impaired meiotic maturation. This phenotype is remarkably similar to that caused by deletion of Dicer, but not to those caused by deletion of Dgcr8 or Drosha, two proteins that also perform essential roles in the miRNA pathway. Thus, both Dicer and Ago2 seem to be required for an endogenous RNAi pathway in mammals whose activity is essential to life. Nevertheless the details of this pathway remain obscure and we still don't know the small-RNAs it relies on, which transcripts it regulates, how its impairment affects embryo development and oocyte maturation, or whether it has a function outside these two processes.To shed light into this poorly characterized pathway my lab will develop an experimental approach and computational pipeline to unbiasedly identify endogenous catalytic targets to Ago2. Aim 3. Define the functions of mammalian. Argonaute proteins in the nucleus In mammals, Argonaute proteins are viewed as exclusively cytoplasmic proteins that associated with P-bodies to fine tune gene expression post-transcriptionally. Yet, our unpublished data as well as studies from other labs suggest that in some cellular contexts a large fraction of Ago seems to be found in the nucleus. Specifically, we found that Ago2 seems to accumulate in the nucleus of resting T cells and move into the cytoplasm upon cell activation. Ago2 has also been reported to localize to the nucleus in differentiated human keratinocytes and when cells go into senescence. While in other eukaryotic organisms the nuclear localization of Argonaute proteins is well studied and recognized, it is largely uncharacterized in mammals and its functional relevance remains unknown. My initial goal is to fully characterize the localization of all four mammalian Argonaute proteins in vivo, to understand under which circumstances these proteins can be nuclear-a fundamental step to understanding which processes require their nuclear localization or how protein localization is regulated.